Experimental determination of thyroid inhibitor residues in foods of animal origin
Experimental determination of thyroid inhibitor residues in foods of animal origin
Thyroid inhibitors (antithyroida gents) are thioureas, which can be categorized into thioredoxins and imidazoles. Most of the early methods of purification treatment use the mercury column method, so that the binding between -SH and mercury occurs, so as to achieve the purpose of purification and enrichment. Source: Food Safety Monitoring Technology (Chemical Industry Press)
Operation method
Gas Chromatography-Mass Spectrometry
Materials and Instruments
Animal tissues Milk Urine samples Move 1. Reagents and materials For more product details, please visit Aladdin Scientific website.
Pentafluorobenzyl bromide N-methyl-N-trimethylsilyltrifluoroacetamide Sodium ethanol Ethanol Silica gel
Solid Phase Extraction Empty Columns Homogenizers Centrifuge Tubes Chromatography Columns
Derivatization reagent 1: Pentafluorobenzyl bromide (PFBBr) (pentafluorobenzyi bromide, 97%).
Derivatization reagent 2: N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) [N- methyl-N-(trimethylsilyl)-trifluoroacetamide, 97%]. Silica gel: 40 to 63 um, 10 nm (100A). Sodium ethoxide/ethanol: weigh 0.2 g NaOH dissolved in anhydrous ethanol and volume to 50 mL. solid phase extraction empty column (6 mL, 10 mL). Internal standard: bis(methylthiouracil) (DMTU).
2. Pre-treatment
Method 1: (Animal Tissue) Grind the animal tissue sample into pulp with homogenizer, weigh 0.5 g in a mortar, add 1 ug/mL methanol solution of DMTU and mix with 2.0 g silica gel, leave it at room temperature for 3 min, then load the column, wash the mortar with rinsing solution and load the column. 10 mL of chloroform rinsing, 6 mL of chloroform containing 30% of methanol (V/V) mixed solvent elution. Blow-dry the eluent, add 0.1 mol/L EtONa-EtOH solution 5000 uL, PFBBr25 uL, and derivatize in a water bath at 25 ℃ for 10 min. pH was adjusted to 3.0 (±0.3) with HCl.3 x 1 mL of dichloromethane was extracted, and the extracts were combined and blown-dry.6 mL of silica gel columns were activated with 5 mL of ethyl acetate and 5 mL of n-hexane in turn; 1 mL of The 6 mL silica gel column was activated with 5 mL ethyl acetate and 5 mL hexane; 1 mL of dichloromethane was used to dissolve the residue; 5 mL of chloroform was used to rinse the column, and the column was eluted with a mixture of 5 mL of dichloromethane-ethyl acetate (1 + 4). Blow dry the eluate, add 50 uL MSTFA, 50 uL dichloromethane. Derivatize at 60 ℃ for 30 min after sealing, and after derivatization, the sample was diluted with n-hexane to 10 mL, and then analyzed by GC-MS.
Method 2 (Animal tissue) 2.0 g of homogenized animal tissue was accurately weighed in a 50 mL centrifuge tube, 50 uL of 1 ug/mL methanol solution of internal standard bis(methylthiouracil) was added, and the extract was extracted by 3x 5 mL of acetonitrile, then the extracts were combined and blown dry. 600 uL of water was used to dissolve the residues, then poured into a mortar, and mixed with 2.0 g of silica gel, and then loaded into the column after being allowed to stand at room temperature for 30 min. 10 mL of chloroform rinsed, and 6 mL of chloroform washed, followed by a 10 mL of chloroform rinsing. The column was eluted with 10 mL of chloroform and 6 mL of chloroform solvent mixture containing 20% methanol. Blow-dry the eluate, add 0.1 mol/L EtONa-EtOH solution 50 uL, PFBBr25 uL, and derivatize in a water bath at 25 ℃ for 10 min. pH was adjusted to 3.0 (±0.3) with HCl, extracted by 3 x 1 mL dichloromethane, and the extracts were combined and blown dry. Add 50 uL MSTFA,5O uL dichloromethane. After sealing, derivatize in a water bath at 60 ℃ for 30 min. After derivatization, the solution was fixed to 1 mL with hexane and left to be analyzed by GC-MS.
Method 3 (Milk and urine samples) Accurately measure 0.6 mL of milk or urine samples into the mortar, add 1 ug/mL of internal standard DMTU methanol solution 50 uL and mix with 2.0 g silica gel, leave it at room temperature for about 30 min and then loaded on a column, washed the mortar with a drenching solution and loaded on the column. The column was drenched with chloroform solution containing 5% methanol (V/V) and eluted with 20% methanol chloroform (V/V) solution, and the eluate was collected. Blow-dry the eluate, add 50 uL of 0.1 mol/L EtONa-EtOH solution, 25 uL of PFBBr, and derivatize in a water bath at 25 ℃ for 10 min. pH was adjusted to 3.0 (±0.3) with HCl, and extracted by 3 x 1 mL of dichloromethane, and the extracts were combined and blown dry. Add 50 uL MSTFA, 50 uL dichloromethane. After sealing, derivatize in a water bath at 60 ℃ for 30 min. After derivatization, the solution was fixed to 1 mL with hexane and left to be analyzed by GC-MS.
3. Instrumental conditions
(1) Chromatographic conditions
Chromatographic column: DB-5MS, 30 m x 0.25 mmx0.25 um;
Chromatographic column program temperature: 100 ℃ constant temperature for 2 min, 15 ℃/min to 260 ℃, 10 ℃/min to 290 ℃, constant temperature for 5 min;
Inlet temperature: 250 ℃;
Injection mode: non-shunt injection, non-shunt time 2 min;
Carrier gas: high purity helium, purity >99.99%;
Inlet volume: 10 L.
(2) Mass spectrometry parameters
Four-stage rod mass spectrometry, E1 ionization source, ionization voltage 70 eV, emission current 150 mA, detection voltage 500 V, ion source temperature 200 ℃; transmission line temperature 250 ℃. Selected ion scan (m/z) methimazole: 241, 261, 275, 294; 2-thiouracil: 360, 365, 380, 381; methyl thiouracil: 374, 379, 394, 395; propyl thiouracil: 402, 407, 422, 423; phenyl thiouracil: 436, 441, 456, 457; the internal standard 4(5. 6)-dimethylthiopyrimidine: 388, 393, 408, 409.